In recent years, it has been increasingly recognized that hepatocytes play important roles in drug discovery. Hepatocytes are indispensable tools for pharmacokinetic studies of drugs, such as prediction of in vivo clearance of drugs or investigation of drug interaction. The molecular mechanisms of various metabolic enzymes and transporters that are expressed in hepatocytes and playing dominant roles in pharmacokinetics (absorption, distribution, metabolism, and excretion) of drugs have been being revealed. Among them, much attention has been paid to the roles of organic anion-transporting polypeptides (OATPs) in the pharmacokinetics of anionic drugs (Non Patent Literature 1).
The OATP family transporters are expressed in various tissues and organs in the body, such as small intestine, kidney, and liver, and among them, OATP1B1 (alias: OATP-C, OATP2, or LST-1), OATP1B3 (alias: OATP-8), and OATP2B1 (alias: OATP-B) are known to be highly expressed in human hepatocytes (Non Patent Literatures 2 and 3). Clinically important drugs such as statins and angiotensin II receptor blockers are known to be taken up by human hepatocytes mainly via OATP1B1, and cyclosporine and rifampicin are known as typical inhibitors of OATP1B1 (Non Patent Literature 2).
It is known that a concomitant use of an OATP1B1 substrate drug with an OATP1B1 inhibitor reduces uptake of the substrate drug into the liver, resulting in increase in the blood level of the substrate drug in clinical settings. Accordingly, evaluation of the drug interaction potential of a test compound via OATP1B1 is very important from the view point of clinical safety. Conventionally, in order to quantitatively evaluate transporter-mediated drug interaction potential of a test compound, an evaluation method (RI method) using a known substrate labeled with a radioisotope is known (Patent Literature 1).
The RI method gives high sensitivity, but is expensive, and also needs caution when handling the samples. Instead, a detection method (fluorescence method), not using any radioisotope, but using a fluorescent compound, is also known. For example, an evaluation method using 6-carboxyfluorescein (6-CFL), which is a good substrate for organic anion transporter 1 (OAT1), has been reported (Non Patent Literature 4).
It can be said that the fluorescence method is easy to operate, is less expensive than the RI method, and has excellent throughput. However, in general, it is difficult to predict whether a compound can or cannot serve as a substrate of a transporter, and, therefore, not many fluorescent compounds that can function as substrates of anion transporters are known. In addition, some conventional fluorescent substrates have disadvantages such as that the detection sensitivity is not necessarily high due to a low fluorescence intensity emitted by the substrate itself, autofluorescence of a test compound, and photobleaching of the fluorescent substrate.